Investigating cellular nanoscale with x-rays from proteins to networks

by Clément Hémonnot
Doctoral thesis
Date of Examination:2016-07-25
Date of issue:2016-08-12
Advisor:Prof. Dr. Sarah Köster
Referee:Prof. Dr. Sarah Köster
Referee:Prof. Dr. Helmut Grubmüller
Persistent Address: http://dx.doi.org/10.53846/goediss-5803

 

 

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Abstract

English

In this work, two macromolecules, both essential components of cells (keratin protein and DNA nucleic acid) were studied by X-ray techniques. I described the implementation and use of different methods to investigate these biopolymers at the nanoscale: solution SAXS, ptychography and scanning X-ray diffraction. Keratin proteins belong to the cytoskeleton of epithelial cells forming a dense network. This network is built from monomers in a stepwise fashion. First, two monomers assemble to form a parallel heterodimer (about 2~nm diameter). Second, tetramers (4 to 5~nm diameter) are formed by an anti-parallel lateral association of two heterodimers, note that at this step the polarity is lost. Third, four tetramers gather together to form a unit-length filament (ULF, 60~nm length and 10~nm diameter). Fourth, under ionic conditions such as 10~mM Tris pH 7.5, ULFs combine longitudinally, undergoing an end-to-end elongation phenomenon resulting in the formation of filaments. Last, under addition of monovalent or divalent ions, filaments bind to form bundles and networks (80~nm diameter). Here, keratin has been studied at three different length scales: i) filaments, ii) bundles and iii) networks. New results were obtained relative to the assembly of tetramers into filaments under different buffer conditions. Solution SAXS revealed distinct structural and organisational characteristics of these filaments. Scanning micro-diffraction was used to study keratin at the bundle scale. Very different morphologies of keratin bundles were observed at different salt conditions. At the network scale, new imaging approaches and analysis were applied to the study of whole cells. Ptychography and scanning nano-diffraction imaging were performed on the same cells, allowing for high resolution in real and reciprocal space, thereby revealing the internal structure of these networks. Lastly, DNA was studied in cells by scanning nano-diffraction, unveiling the compaction of DNA during the cell cycle. In this chapter, the most important findings presented in the different parts of this work and their significance are summarised.
Keywords: Small-angle X-ray scattering; Scanning X-ray diffraction; Biological Cells; Keratin protein; DNA compaction
 
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